Program Director/Principal investigator (Last, First, Middle): Dustln, Michael, Lotan PROJECT SUMMARY (See instmctions): The long-tenn goal of our work is to determine the mechanisms by which conventional and regulatory T lymphocytes are activated by very small numbers of MHC-peptide complexes on the surface of antigen presenting cells. This sensitivity determines the threshold for activation of mature T cells and thus may play an important role in tolerance and immunity. Work from our lab and others has shown that antigen receptors, adhesion molecules and cytoskeletal dynamics collaborate to form a highly ordered immunological synapse, which sustains signaling by unknown mechanisms. We have observed that the organization and dynamics of T cell antigen receptors in the immunological synapse depends upon which adhesion systems are engaged. A prominent feature of the immunological synapse fomned with different adhesion systems is small TOR clusters in the periphery of the contact area. In Aim 1 we will test the role of peripheral TOR clusters in sustained signaling and T cell activation. We will identify an optimal combination of iCAM-1, CD48, CD80 and MHC-peptide complexes in supported planar bilayers and will compare this to what is observed with a professional antigen presenting cell type, the dendritic cell (DC). Co-receptor molecule CD4 plays an important role in setting T cell sensitivity to antigen. In Aim 2 we will test the importance of different putative interactions of CCM with MHC class II, Lck, membrane domains, the TCR and itself in T cell sensitivity to antigen. The actin cytoskeieton has an essential role in T cell responses to antigen. Cofilin, cortactin and Arp2/3 are associated with dynamic lamellipodia, which are sites of sensitive signal initiation. The lamella is directly behind the lamellipodium and contains actin filaments that are stabilized by tropomyosin and the actin-integrin adapter protein talin. In Aim 3 we will investigate the actin based structures in immunological synapses formed with different adhesion systems. Then we will use RNA interference and overexpression studies to manipulate the expression of actin regulators. In the context of regulatory T cells, we will study the role of intermediate filament proteins. These studies will lead to a greater understanding of sensitivity to self and foreign antigen in immune response and regulation. RELEVANCE (See instructions): The ability of conventional T lymphocytes to respond to foreign antigens with high sensitivity is essential to vaccination, whereas the ability of regulatory T cells to sensitively respond to self antigens presents autoimmunity. Understanding the fundamental structures that mediate sensitive antigen recognition may lead to better immunotherapies for cancer and infectious diseases and treatments for autoimmune diseases.